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Bone remodeling: an operational process ensuring survival and

bone mechanical competence
1 1 1✉
Simona Bolamperti , Isabella Villa and Alessandro Rubinacci

Bone remodeling replaces old and damaged bone with new bone through a sequence of cellular events occurring on the same
surface without any change in bone shape. It was initially thought that the basic multicellular unit (BMU) responsible for bone
remodeling consists of osteoclasts and osteoblasts functioning through a hierarchical sequence of events organized into distinct
stages. However, recent discoveries have indicated that all bone cells participate in BMU formation by interacting both
simultaneously and at different differentiation stages with their progenitors, other cells, and bone matrix constituents. Therefore,
bone remodeling is currently considered a physiological outcome of continuous cellular operational processes optimized to
confer a survival advantage. Bone remodeling defines the primary activities that BMUs need to perform to renew successfully
bone structural units. Hence, this review summarizes the current understanding of bone remodeling and future research
directions with the aim of providing a clinically relevant biological background with which to identify targets for therapeutic
strategies in osteoporosis.

Bone Research (2022)10:48

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; https://doi.org/10.1038/s41413-022-00219-8

INTRODUCTION BONE MODELING, REMODELING, AND THE MUTUAL

Despite the tremendous efforts of researchers studying bone
remodeling for more than 50 years, the intrinsic spatial,
biomolecular, and mechanotransduction complexities in bone
remodeling continue to be debated. Initially, a two-stage
process involving two types of cellular machinery was
considered to be responsible for bone remodeling, in which
bone formation by osteoblasts follows bone resorption by
osteoclasts to achieve net bone mass equilibrium upon
physiological maturity.1 However, recently identified cellular
events, coordination pathways and anatomical structures have
allowed a better understanding of the genesis, differentiation,
activity, crosstalk and death of the entire cell population
involved in bone remodeling.2 These discoveries have shed
new light on the operational processes ensuring bone mass
renewal without bone mass loss under physiological condi-
tions. Bone remodeling is activated by local and systemic
factors, supporting the concept that targeted remodeling is
activated by local factors and that stochastic remodeling is
activated by systemic factors, with these factors cooperating to
maintain mechanical competence and meet concurrent meta-
bolic demands. This new information implies that the determi-
nants of the focal balance in bone mass after remodeling are
the integrated effects of both mechanical and metabolic
environmental conditions.
This review provides a novel integrated picture of the
operational bone remodeling processes by describing, updat-
ing, and reexamining the current evidence and its biological
plausibility. In this review, bone remodeling is described
as a continuous flow of cellular signaling and connected
events, not as a process comprising stages, as it has been
historically presented.
REGULATION OF BONE RESORPTION AND FORMATION
In vertebrates, bone modeling and remodeling are essential
processes that are activated throughout life and are regulated by
distinct temporal cellular constituents that ensure functional bone
adaptation and vertebrate survival. Bone modeling adapts bone
shape to variable mechanical demands during growth and aging
through cellular events that determine bone resorption and
formation on opposing cortical and cancellous surfaces. This
implies the existence of a modeling drift, which moves a bone
structural unit over time in the direction defined by growth
patterns, and adjusts the bone mass distribution to the stresses
and strains induced by locomotion and physical activity.3 By bone
remodeling, old or damaged bone is replaced with new bone
through a sequence of cellular events occurring on the same
surface without any change in bone shape.4
In the 1960s, Frost5 recognized that a forming osteon in
mammalian compact bone consists of a group of synchronous
cells, suggesting “control mechanisms which are functionally and
temporally ordered, discontinuous and discrete”. Frost advanced
the enlightening notion of a basic multicellular unit (BMU) as a
transient anatomic structure in bone remodeling and introduced
the quantum concept of bone remodeling, which is analogous to
quantum theory in physics. Quantum physics explains the property
of matter at the smallest scale. It defines the behavior of the
minimum, discrete amount, i.e., the quantum, of any physical
entity on the assumption that all phenomena in a submicroscopic
system exhibit quantization.6 In an analogy, Frost intuitively
defined a quantum of bone remodeling as a discrete change in
bone mass resulting from the coordinated activity of an individual
BMU at an anatomically discrete locus.1 The quantum concept
conceived by Frost and later extended by Parfitt7 has had

1
Osteoporosis and Bone and Mineral Metabolism Unit, IRCCS San Raffaele Hospital, Via Olgettina 60, 20132 Milano, Italy
Correspondence: Alessandro Rubinacci (rubinacci.alessandro@hsr.it)

Received: 2 August 2021 Revised: 2 May 2022 Accepted: 15 May 2022

© The Author(s) 2022

 The operational process of bone remodeling
S Bolamperti et al.
2
Direction of BMU
propagation
Symmetry Nerve
line
id
teo
Os
The cutting Reversal
cone zone
Reversal cell Osteoclast
Osteoprogenitors Monocyte/
macrophage
Fig. 1 The cutting cone. The cutting cone originates in close proximity to neurovascular axial bundles and is generated by the propagation of
the basic multicellular unit (BMU). The cutting cone consists of a set of osteoclasts, followed by a set of osteoblasts, reversal cells and
secondary osteoclasts that cover the so-called reversal zone. At the end of the reversal zone, a set of osteocytes generate the closing zone. A
line of symmetry divides in half the representation of a complete BMU in the cortex moving toward the longitudinal axis of the long bone.
One-half of cortical BMU is similar to the BMU at the cancellous surface, although in cancellous bone, the BMU is separated from the marrow
by a specific cell structure called the canopy
profound implications for the understanding of all aspects of bone
pharmacology and physiopathology,8 particularly in the osteo-
porotic context.9 A remarkable idea of the early sixties that is in
line with the modern theory that quantum biological phenomena
can lead to evolutionary advantages.10
Frost recognized that the change in bone mass caused by the
focal balance in each remodeling cycle of resorbed bone and
formed bone is not an outcome of isolated “working” cell packets
but is derived from the interacting parts of the whole BMU, which
endures longer (9 months) than the lifespan of each single
component.4 This implies a continuous and ordered cell supply
that depends on the division frequency of each progenitor cell
and the lifespan of each differentiated cell. A tightly maintained
equilibrium between genesis and apoptosis is therefore critical for
a properly functioning BMU.11 Recent advances have added
complexity to the original BMU description. The number of cells
considered to constitute a BMU has expanded to include all bone
cells at all differentiation stages, interacting with their progenitors,
T cells and bone matrix components. The BMU includes, in
particular, a set of osteoclasts localized in the “cutting cone”
followed by a set of cells, including reversal cells and osteoblasts,
localized at the reversal zone; and a set of osteocytes localized in
the closing zone. These cell sets constitute a secondary osteon
around the neurovascular bundle wich is axially located with the
connective matrix in the Haversian canal. A visualization of a
complete BMU can be acquired only with a longitudinal section of
cortical bone, where Haversian canals run parallel to the sectional
plane (Fig. 1). Notably, the 3D trabecular network prevents the
proper visualization of a complete BMU in cancellous bone.12
In bone remodeling, osteoblast–osteoclast interactions are
necessary and must be coordinated in time and space to maintain
the focal bone remodeling balance; this balance refers to the net
amount of tissue resorbed and formed at each remodeling site
(i.e., focal point) to maintain the structural integrity of the tissue.
At maturity, the focal balance (neutral) in bone remodeling is
determined by equal bone aliquots, which are removed and
synthetized. In other periods in life or as a consequence of
metabolic diseases, the focal balance can be negative (bone mass
is lost) or positive (bone mass is gained). Focal coupling in bone
remodeling strictly refers to the transfer of the information within
the cell pool responsible for bone remodeling. This operational
Cortical
A similar
remodeling
process occurs
in cancellous
bone, but
within a canopy
Closing
zone
Osteocyte Pericyte
Active
osteoblast
process couples the events necessary to remove and replace bone
units under a hierarchy of time and space, independent of their
focal balance; in fact, focal remodeling imbalance does not imply
“uncoupling”. At each remodeling site, coupling indicates that the
receptors on the osteoclast membrane as well as the regulatory
factors released by osteoclasts are coupled with the sequential
recruitment and differentiation of osteoblast lineage cells toward
the mature phenotype, laying down the bone matrix and inducing
mineralization. Coupling therefore implies that a commensurate
change in bone formation follows any pathology- or therapy-
related modification of bone resorption.
At each remodeling site, coupling is asynchronous: at any given
time, bone is being resorbed at some sites, while it is being formed
or is in the reversal phase from bone resorption to bone formation
at other site. This implies that there is continuous focal transient
loss of bone that is fully reversed in balanced remodeling; as the
number of activated remodeling sites increases, the transient loss
of bone, which is defined as the “virtual space” of bone remodeling,
increases. As coupling takes place in different locations at different
times, it requires local regulatory factors to transfer information
among cells and tissue locations according to bone remodeling
needs. Therefore, remodeling is defined as a dynamic physiological
process executed through the coupled activity of osteoclasts and
osteoblasts belonging to a BMU. Conversely, bone turnover is the
outcome of bone remodeling that defines the amount of bone
removed and formed within a given volume in a given time and is
determined by the number of BMUs and by the focal balance
within each BMU. As Parfitt said “Bone turnover refers to proportional
bone volume replacement per unit time, and is usually expressed as
percent/year […]. If bone turnover is 10%/year, then the mean lifetime
of each moiety of bone is 10 years”.13
OSTEOCYTES: TUNING, INITIATION AND TERMINATION OF
BONE REMODELING
Osteocytes are a relatively permanent phenotype of bone cells that
Frost estimated to have a lifespan of ~25 years1 and play a master
role in bone remodeling. Osteocytes represent more than 90% of
bone cells in the adult skeleton14 with a average cell density highly
dependent on the specie, bone anatomical site, and subiect
analyzed.15 Recent studies performed using new imaging
Bone Research (2022)10:48

techniques have estimated that 19 000–28 500 cells per mm3
populate the human skeleton, accounting for ~42 billion individual
cells.16 Osteocytes consist of a stellate body connected by slender
cell processes (50–100 processes per cell). The asymmetrical
“arborization” of dendrites polarizes toward bone surfaces, where
the dendrites contact osteoblasts undergoing growth or remodel-
ing or, during bone surface quiescence, bone lining cells. The term
“arborization” was originally introduced by Marotti and Palumbo,17
and it now refers to “dendrogenesis”, the asymmetric and
asynchronous formation of osteocyte dendrites. Short radiating
dendrites extend toward mineralized surface, and long radiating
dendrites extend toward the vasculature during the progressive
translocation of cell bodies farther from the vascular surface due to
the secretion of the osteoblastic lamina. Osteocytogenesis and
dendrogenesis are discussed in a subsequent section (“The
osteoblast pool: recruitment, expansion and osteocytogenesis”).
An adult human skeleton includes 23 trillion osteocyte
connections with each other and with bone surface cells.16
These connections form a 3D protoplasmic network that
constitutes a matrix-integrated functional syncytium, which does
not cross cement lines but does establish direct contact with the
bone marrow, resides in low-oxygen microenvironment and
comprises multiple elements. During aging in both humans and
rodents, this network deteriorates. Throughout aging, a large
and linear reduction in dendrite number and cell body density
directly related to the deterioration of cortical parameters has
been observed.18 Since decreased osteocyte number accom-
panies reduced dendritic density, it has been suggested that
dendrite loss might contribute to diminished osteocyte viability
because a certain degree of locally and/or systemically triggered
anabolic signaling through the osteocyte-lacunocanalicular
system is lost (see below).18
The osteocyte-bone lining cell syncytium displays gap junctions
(connexins) that allow the transfer of information between cells.
Connexin43 (Cx43) is the most abundant connexin in osteocytes,
and global knockout of Cx43 expression is lethal at birth.19
Conditional knockout of Cx43 in osteoblasts and osteocytes in
mice led to various degrees of osteopenia depending on the
differentiation state in which the deletion was induced,20,21
suggesting that functional Cx43 in osteoblasts and osteocytes is
essential for normal bone mass acquisition and maintenance.
The osteocyte-bone lining cell syncytium is endowed within the
lacunocanalicular network of cavities filled with bone extracellular
fluid (BEF). BEF has a different ionic composition from systemic
extracellular fluid (SEF)22 and establishes an extensive contact
surface (215 m2, which is 120-fold the size of the trabecular
network) with the mineralized matrix to allow efficient, short-term
mineral exchange with SEF.23–26 This specific physical environ-
ment allows osteocytes to govern metabolic demands and
mechanotransduction for bone mass adaptation.27 The strain-
induced flow of BEF in the lacuno-canalicular network exerts a
shear stress (fluid flow shear stress, FFSS) on osteocyte bodies,
which undergo dendrogenesis to activate several classes of
mechano-sensors regulating specific gene expression patterns.28
Osteocytes might sense FFSS through 1. “collagen hillocks”,
which are collagen matrix projections in osteocyte canaliculi that
directly link the matrix to osteocyte dendrites;29 2. β3 and β1
integrins, which participate to focal adhesion kinase (FAK)
complex formation in osteocyte dendritic projections;30 3. primary
cilia, which have a flow-sensing function that leads to increased
osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-
B ligand (RANKL) ratio via a calcium-independent mechanism;31 4.
connexin43, a component of gap junctions that mediates the
transduction of mechanical signals;32 and 5. mechanosensitive ion
channels, such as those composed of Piezo1, which are highly
sensitive to osteocyte membrane tension.33
The deformation of the osteocyte cytoskeleton elicits Ca2+
influx signaling via the activation of TRPV4.34 The generation of
Bone Research (2022)10:48
The operational process of bone remodeling
S Bolamperti et al.
3
Ca2+-dependent contractions of the cell membrane favors the
production and release of extracellular vesicles (EVs) containing
bone regulatory proteins.35 EVs are ubiquitous lipidic organelles
that mediate the intercellular transfer of information through their
cargo, which includes both proteins and nucleic acids.36 In
osteocytes, EVs can be observed in proximity of the osteocytic
network.37 In response to mechanical stimuli and subsequent
Ca2+ influx, osteocyte lines have been observed to release EVs
containing RANKL, OPG, and sclerostin.35
All the mechanical signals received by the osteocyte-bone
lining cell syncytium modulate cell apoptosis and survival as well
as the anabolic Wnt pathway in bone. Secreted Wnt protein
stimulates target cell via the β-catenin-mediated (canonical) and
β-catenin-independent (noncanonical) pathways. The canonical
pathway has emerged as the predominant component of Wnt
signaling in bone, positively affecting the entire osteoblast
lineage.38 Wnt proteins bind to their receptors (Frizzled) and
coreceptors (LRP5/6) to promote the stabilization of β-catenin in
the cytoplasm, which translocates to the nucleus, where it induces
the expression of osteogenesis-related genes.38,39 Many studies
have demonstrated that β-catenin is required for bone formation
and is activated during multiple stages of osteoblast differentia-
tion to regulate both osteoblast and osteoclast.40–43 In fact, WNT–
β-catenin signaling in osteoblasts and osteocytes indirectly
represses osteoclast differentiation and bone resorption by
stimulating the secretion of OPG.38
Mechanical signals activate the anabolic Wnt pathway in bone
through the suppression of the Wnt receptor antagonist sclerostin
(SOST). Recently, Sato et al.20,44 showed that FFSS induces the
disruption of the FAK-integrin complex, thereby inhibiting histone
deacetylase 4/5 (HDAC4/5), a negative regulator of SOST expres-
sion in osteocytes45 modulated by parathyroid hormone (PTH)
treatment.46 Osteocytes can also decrease SOST expression after
sensing decreases in oxygen levels.47
In vivo anatomical studies have suggested that the presence of
apoptotic osteocytes at microdamaged sites correlates with the
recruitment of osteoclasts at microcracks.48 In addition to their
fundamental role as mechanosensors, osteocytes appear to be
spatially, temporally and mechanistically linked to bone remodel-
ing activation, particularly by regulating RANKL (as is discussed
below). RANKL is routinely found as a membrane bound protein
(mRANKL), but it can also be cleaved into a soluble form
(sRANKL)49 or delivered via EVs.35 RANKL binds the receptor
activator of nuclear factor kappa-B (RANK) on osteoclasts to
induce osteoclastogenesis.50
In 2011, Nakashima et al.51 showed that the conditional deletion
of RANKL expression in osteocytes using a DMP1 promoter caused
a severe osteopetrotic phenotype. This osteopetrotic phenotype
was confirmed by Xiong et al.,52 who in addition found that RANKL
mRNA, isolated from total bone of DMP1-cre+, RANKLloxp/loxP mice,
showed very little variation compared to that of wild type mice.
The concentration of circulating sRANKL was unaltered compared
to that in wild type animals. However, a 70% reduction in
osteoclast number in the cancellous bone of these transgenic
mice highlighted the relevance of local RANKL production.52 As
the activation of Dmp1-Cre promoters is not exclusive to
osteocytes,53 the conditional deletion of RANKL expression was
performed under the control of the more specific SOST promoter.
This conditional deletion generated a phenotype resembling the
previous one, with a significant decrease in osteoclasts number in
cancellous bone.54 Taken together, these in vivo studies confirmed
that osteocytes produce RANKL and that osteocytic RANKL is
fundamental to sustaining bone remodeling.
Whether osteocyte RANKL is soluble, is transferred via vesicles
or is membrane-bound is not fully clear. Bonewald et al.14 first
demonstrated that the MLO-Y4 osteocyte line expresses RANKL at
the cell surface and at dendritic processes. MLO-Y4 cells and
primary murine osteocytes cocultured with bone marrow
 The operational process of bone remodeling
S Bolamperti et al.
4
Bone marrow Osteoclast precursor
RANK
RANK
RANKL
RANKL
ATP
P2Y2
ATP
P2Y2
P2X7
ATP
Bcl-2 Panx1
Mic
roc
rac
k osteocyte
ATP
P2Y2
P2X7
Extracellular Panx1
matrix
Fig. 2 The “find me” message from apoptotic osteocytes. Under fatigue failure, osteocytes undergo apoptosis, whereas the osteocytes
surrounding the apoptotic osteocytes, the “bystander osteocytes”, show upregulation of the antiapoptotic protein BcL-2, which protects them
from death. Apoptotic osteocytes activate the Panx1/P2XR pathway to induce the release of ATP into the extracellular compartment as a
specific “find me” signal. ATP binds P2Y2 receptors on bystander osteocytes, which in turn produce and release RANKL at the interface with
the bone marrow. RANKL promotes the recruitment of osteoclast progenitors and osteoclastogenesis
precursors supported osteoclastogenesis, but their conditioned
media alone did not have a similar effect,55 suggesting that
cell–cell interactions between osteocytes and osteoclasts are
needed for osteoclastogenesis. In a different study, the condi-
tioned media of apoptotic MLO-Y4 cells increased osteoclast
formation, osteoclast size and osteoclast precursor migration.
Interestingly, in apoptotic MLO-Y4 cells, both RANKL mRNA and
protein expression were upregulated, suggesting that the condi-
tioned media of this cell culture might contain sRANKL.56 On the
other hand, the conditioned media of a non apoptotic MLO-Y4
culture did not alter any of these aforementioned cell para-
meters.56 In another in vitro study, primary osteocytes were
cocultured on a 3D collagen scaffold with osteoclast precursors
separated by a porous membrane. Again, the osteocytes
supported osteoclast formation, but the efficiency of their effect
decreased with a decrease in the pore size of the membrane.
Confocal microscopy showed that large pore membranes allow
osteocyte dendrites to touch osteoclast precursors through the
pores, but this contact was not observed when small-pore
membranes were used.57 Therefore, the authors of this study
hypothesized that under specific conditions, such as when
osteocytes cannot physically touch OC precursors, osteocytes
might support osteoclastogenesis by secreting EVs containing
RANKL.58 Distinguish the exact localization and form of RANKL
produced by osteocytes in vivo is particularly complicated, as the
antibodies used for immunohistochemical analyses do not
distinguish between the soluble and membrane forms. Therefore,
to evaluate the form of RANKL that induces osteoclastogenesis, a
mouse model was generated with a sheddase-resistant form of
RANKL with no detectable sRANKL levels in the circulation.
During growth, this lack of sRANKL did not cause bone mass
alteration, but in the adult model mice, the number of osteoclasts
was reduced with an increase in cancellous bone mass.59
However, mice lacking sRANKL expression and ovariectomized
still displayed bone loss because of the lack of estrogen, indicating
that, although mRANKL can support most RANKL functions, the
RANKL
Osteoclast
RANK
Bcl-2
P2Y2
ATP
P2X7
Bcl-2 Panx1
By-stander
Apoptotic
osteocyte
soluble form is necessary to sustain bone remodeling under
certain conditions.59 In support of this hypothesis, mice over-
expressing sRANKL only in the liver, and therefore also in blood
circulation, displayed a significant decrease in bone mass with an
increase in the number of osteoclasts on cancellous bone surfaces
during aging.60
In vivo, osteocyte apoptosis is fundamental for initiating bone
remodeling,61 independent of the stimulus causing osteocyte
death such as estrogen loss,62 fatigue,63 or unloading.64 An in vivo
immunohistochemical analysis showed that the RANKL signal in
bone tissue was stronger in an area around 150–200 μm from the
apopototc osteocytes.65 Interestingly, inhibiting apoptosis pre-
vented the RANKL production by neighboring osteocytes,
suggesting that “bone remodeling follows a common paradigm
for localized tissue repair”, with different response-induced factor
released from apoptotic osteocytes near the damage site and
“bystander” osteocytes.63 MLO-Y4 cell apoptosis generated ATP
release from cells via Pannexin 1 channels (PANX1) opening.66
The same phenotype was observed in mice treated with an
inhibitor of the P2X7 receptor (P2X7R), which is a coactivator
of PANX1.67 In periodontal ligament cells, ATP induced the
upregulation of RANKL expression via a P2Y1 receptor-
cyclooxygenase-dependent pathway.68 Since the release of ATP
from apoptotic cells functions as a specific “find me” message for
phagocytic cells,69 ATP release during osteocyte apoptosis might
be a specific “find me” signal directed to neighboring osteocytes.
Indeed, ATP from apoptotic osteocytes might bind P2Y2 receptors
expressed on bystander osteocytes, which in turn might activate
RANKL production and release67 (Fig. 2). Moreover, bystander
osteocytes (in 1–2-mm proximity to the focal damage site)
expressed the antiapoptotic protein BcL-2. This defense mechan-
ism might be necessary to prevent osteoclastogenic signal
damaging viable osteocytes70 (Fig. 2).
As osteocytes are deeply embedded in the bone matrix,
osteocyte apoptosis or autophagic death causes secondary
necrosis due to inhibited phagocytosis by scavenger cells.
Bone Research (2022)10:48

Table 1. Systemic and local factors influencing osteocyte function
Stimuli Features affected
(Systemic/Local) (Local)
PTH Life-Span Sclerostin, DKK1, FGF23, DMP1, Phex, MEPE,
Estrogens Network formation Sema 3A RANKL, OPG SASPs, TGF-β1 OCN
1,25(OH)2D3 Secretome
Irisin Mechanotrasduction
Calcitonin
Senescence
TGF-β1
Pi
pO2
Major systemic and local stimuli that influence osteocyte activity, the functions affected, the factors released upon exposure to the stimuli, and the impact on
the system
Necrotic osteocytes release damage-associated molecular pattern
(DAMP) proteins after the cell membrane is disrupted.61 DAMP
proteins flow through the canalicular network and reach the
bone/marrow interface, where they bind to pattern recognition
receptors (PRRs) on bone marrow cells.71 When activated by
DAMP–PRR binding, monocytes and macrophages produce
proinflammatory cytokines, such as TNFα, IL-6, and IL-1.72 These
cytokines in turn stimulate the expression of RANKL in osteo-
blasts,73 enhancing the osteoclastogenesis previously activated by
the release of ATP by apoptotic osteocytes.66 DAMPs can directly
regulate osteoclast formation by activating the membrane-bound
C-type lectin receptor Mincle. Mincle activation triggers calcium
signaling and oxidative phosphorylation in osteoclasts, inducing
osteoclast differentiation.74
Under physiological conditions, osteocytes exert an inhibitory
effect on osteoclasts by contributing to the osteoblast production
of OPG via Wnt signaling.75,38 OPG functions as a soluble decoy
receptor that binds RANKL, preventing its interaction with RANK
expressed by osteoclast progenitors. The major cell source of OPG
is still debated, as it depends on the bone compartment and on
the age of the subject considered. In addition to osteocytes, other
cells can be sources of OPG or RANKL; these cells are
osteoprogenitors, osteoblasts49 or cells of the immune system
(B and T cells),76 which are all simultaneously present in a BMU.
Nevertheless, OPG released by mature osteoblasts and/or
osteocytes may constitute the termination signal of the resorp-
tion phase in the remodeling cycle.77
In addition to microcrack-induced signals to activate the healing
of microdamage foci through targeted remodeling, osteocytes
sense systemic and local stimuli that affect their functions and
survival. When exposed to a stimulus, osteocytes release several
factors affecting (1) bone remodeling at stochastic loci, (2) matrix
mineralization, (3) lacunocanalicular remodeling, (4) mineral
homeostasis, and (5) fat metabolism and myogenesis (Table 1).
PTH/PTH-related peptide (PTHrP)
Under physiological conditions, osteocytes integrate the
responses of bone to mechanical loading and PTH, since the
mechanotransduction process requires PTH receptor 1 (PTHR1)
activation to downregulate SOST expression. SOST expression
downregulation positively fine-tunes the bone remodeling bal-
ance by promoting osteoblast expansion.78 When PTHR1 on
osteocytes is activated by chronically high PTH concentrations or
when PTHR1 is constitutively active, osteocytes initiate bone
remodeling by expanding the osteoclast pool through the release
of RANKL,79 particularly within the bone cortex.80 In contrast,
when PTHR1 is deleted, RANKL release and subsequent osteo-
clastogenesis do not occur.81
Interestingly, in addition to BMU activation, three hours after
PTH exposure, demineralization of the bone matrix surrounding
Bone Research (2022)10:48
The operational process of bone remodeling
S Bolamperti et al.
5
Factors released Final outcome
(Local/Systemic) (Systemic/Local)
• Bone Remodeling Balance
• Matrix Mineralization
• Perilacunocanalicular Remodeling
• Errors correction in Ca2+/P homeostasis
• Energy metabolism
• Myogenesis
• Oxidative stress
osteocytes accompanied by compensatory mineral deposition
has been observed.82 The same outcome was observed in cases
of excess PTH concentrations both in rats83 and humans84 and
under PTH modulation after exercise.85 This process is mediated
by osteocytes and is called perilacunar remodeling (i.e.,
“osteocytic osteolysis”); it was initially recognized during lacta-
tion. Perilacunar remodeling has been associated with the
activation of PTH-related peptide (PTHrP)–PTHR1 signaling, which
induces matrix resorptive activity in osteocytes similar to that in
osteoclasts.86 Perilacunar remodeling integrated with bone
remodeling contributes to the maintenance of bone quality.
Perilacunar remodeling is an additional component of the long-
term error correction mechanism to maintain plasma calcium
levels, in addition to the homeostatic regulatory mechanism in
osteoclasts (RANKL-mediated). The efficiency of this additional
and independent component of bone remodeling in calcium
homeostasis under PTH regulation remains unclear because of
the lack of a relationship between serum PTH and osteocyte
lacuna characteristics.87
Nevertheless, as has been recently discussed,88 osteocytes per
sé function independently of associated endocrine loops and
exhibit an evolutionary advantage in mineral homeostasis, which
is particularly highlighted in the rapid minute-to-minute regula-
tion of the BEF-[Ca2+] in teleosts,89 as well as in mammals.26,90
Osteocytes, through PTHR1 activation, might counteract age-
related bone mass loss. In fact, during aging, as well as under the
action of several stressors [see Farr et al.91 for a review], senescent
osteocytes accumulate in the bone microenvironment and acquire
a distinctive proinflammatory secretome, termed the senescence-
associated secretory phenotype (SASP),92 which leads to imbal-
anced bone remodeling with increased resorption and decreased
formation. When PTHR1 was deleted in mouse osteocytes in vivo
(Dmp1-PPRKO), the affected mice displayed age-dependent
osteopenia related to a decrease in osteoblast activity with a
parallel rise in osteoclast number and activity. The imbalanced
bone remodeling in these animals was partially due to a
sclerostin-dependent decrease in osteoblast activity and the lack
of osteocyte protection from oxidative stress.93
Estrogen
Osteocytes respond to estrogen by producing the protein
semaphorin 3A (Sema3A). Sema3A released into the bone
microenvironment binds to its receptor on osteocytes and
promotes their survival. The autocrine loop initiated via Sema3A
is mainly triggered in the mature cell stage, which is the dominant
characteristic of cells in the adult skeleton and is critical in bone
remodeling balance, counteracting age-related bone loss. Auto-
crine loop impairment caused by estrogen deficiency induces an
osteoporosis phenotype.94 In addition to its effects on bone
remodeling, estrogen regulates mechanotransduction in osteocytes
 The operational process of bone remodeling
S Bolamperti et al.
6
a
Bone marrow envelope

Pericyte
Bone lining cells
Can
opy

Osteoblast
Monocyte / Macrophage precursor
Microcrack
Primary Remodeling

ts
Osteomorph

as
osteoclast compartment

bl
eo
st
O
Secondary
Re osteoclast
so

id
rb

eo
ed

st
EC

O
M
Reversal cells, RvCs

b Bone marrow envelope

Pericyte Can
opy
Bone lining cells

1. Glucocorticoids
Monocyte/Macrophage 2. Myeloma
Microcrack 3. PostM osteoporosis
Primary 4. Alendronate
osteoclast Osteomorph 5. Aging

Secondary
osteoclast No transition to osteoblasts
Re
so
rb
ed

Reversal phase arrest

EC

Reversal cells
M

Fig. 3 The bone remodeling compartment (BRC). a The BRC provides the correct microenvironment to link bone formation and resorption
through local signaling. The bone marrow envelope (BME) is a layer of cells of mesenchymal origin and a reservoir for osteoprogenitors that
covers the layer of bone lining cells (BLCs). Once remodeling is initiated, osteoclasts lift the BME from the BLC, inducing BME cells to form a
structure called the canopy. The canopy separates the remodeling site from the remainder of the bone marrow to allow osteoclast and
osteoblast precursors to enter the blood compartment. After resorption, osteoclasts either undergo apoptosis or dedifferentiate into
osteomorphs. The resorbed surface is then colonized by secondary osteoclasts and reversal cells. Reversal cells are osteoblast progenitors that
digest fibrillar collagen remnants, similar to BLCs. Secondary osteoclasts and reversal cells provide the basis for the recruitment and expansion
of osteoblastic pools (b) except under certain counteracting conditions, such as glucocorticoid or alendronate treatment, myeloma, or
postmenopausal osteoporosis

by affecting FFSS,95 Wnt/β-catenin expression,96 and Cx43 expres- 1,25(OH)2D3

sion.97 Its removal triggers osteocyte apoptosis and alters the
oxidative microenvironment, leading to the loss of osteocyte
resistance to oxidative stress.
TGF-β1
Recently, osteocyte-intrinsic TGF-β1 signaling was discovered as a
regulator of lacuno-canalicular remodeling. The suppression of
TGF-β1 signaling in osteocytes, either pharmacologically (by TGF-β
receptor type I kinase inhibitors) or genetically (by specific deletion
of the receptor TβRII in DMP1-cre mice), leads to the reduction
osteocyte dendrites length and total lacuno-canalicular area. The
deterioration of the lacuno-canalicular network was accompanied
by the decrease in the gene expression of Sost, and metallopro-
teases Mmp2, 13, 14, which are involved in the lacuno-canalicular
remodeling, and by the reduction of fracture resistance in cortical
bone despite no differences in cortical thickness and geometry,
and increase in trabecular bone mass, likely due to the observed
decrease in osteoclasts number and surface.98 The observation that
lacuno-canalicular remodeling is an essential component of bone
mechanical competence sustains the view that osteocytes play an
evolutionarily conserved role in bone quality control.98
In addition to its systemic effects, 1,25(OH)2D3 binds to VDR in
osteocytes in an autocrine manner, and as they mature,
osteocytes acquire the capacity to convert physiological levels of
25(OH)D to 1,25(OH)2D3.99 The exact role of 1,25(OH)2D3 in
osteocyte metabolism is still uncharacterized. However, recent
observations have pointed out that 1,25(OH)2D3 a) stimulates the
production of fibroblast factor 23 (FGF23) in osteocyte-like cells;100
the hormone FGF23 is primarily involved in phosphate home-
ostasis and vitamin D synthesis; b) modulates matrix mineraliza-
tion by downregulating dentin matrix protein-1 (DMP-1);101 and c)
regulates osteocyte perilacunar remodeling and canalicular
organization through the activation of matrix resorption genes.102
Phosphate
The establishment of X-linked hypophosphatemia (XLH) mouse
models, characterized by elevated serum FGF23 levels, which
caused decreased 1,25(OH)2D3 levels and hypophosphatemia, led
to the discovery of the role played by phosphate in osteocyte
functioning, which is triggered in response to disruption of plasma
phosphate homeostasis.103 How osteocytes sense phosphate
levels and subsequently regulate perilacunar remodeling103 and

Bone Research (2022)10:48


FGF23 and 1,25(OH)2D3 synthesis104 is still unclear. However, as
has been previously discussed,105 the phosphate-sensing mechan-
ism in osteocytes may involve the activation of FGFR1 and high-
affinity Na+-Pi cotransporters.
Calcitonin
Osteocytes express specific calcitonin receptors, which are
progressively lost with age.106 Calcitonin can potentially modify
the osteocyte production of sclerostin and FGF23.107
Irisin
Irisin, a myokine produced by muscles, reduces osteocyte
apoptosis and increases osteocyte number.108 These effects
suggest a regulatory loop involving osteocytes and muscle
metabolism, particularly since osteocytes inhibit skeletal muscle
differentiation by producing a large number of cytokines, which
might permeate the periosteum or diffuse into the circulation to
negatively regulate myogenesis109.
These findings indicate that osteocytes can be considered the
“social” coordinators of bone cells because they are responsible
for maintaining bone physiological responsiveness to mechanical
and metabolic demands. When osteocytic coordination fails,
severe osteoporosis develops.110
BONE LINING CELLS (BLCS), CANOPY CELLS AND PERICYTES:
COMPOSITION OF THE VASCULAR BONE REMODELING
COMPARTMENT (BRC)
BLCs, derived from the final differentiation of osteoblasts, are
located on quiescent trabecular, endosteal, and endocortical
surfaces at the end of bone formation. BLCs are a cell population
that differs from both marrow cells111 and osteoblasts. BLCs are
flattened and exibits lower synthetic activity, with little cytoplasm
or endoplasmic reticulum,112 but they retain a social attitude.
Similar to osteocytes, BLCs express intercellular adhesion
molecule-1 (ICAM-1) to maintain functional contact with osteo-
blasts, osteoclast precursors113 and mature osteoclasts.114 They
cover quiescent bone surfaces that are not undergoing remodel-
ing, but they are not “quiescent” as the older literature suggests.
Although their specific function has not yet been fully defined,
BLCs might form an epithelial-like membrane that functions as an
ion partition system between bone and systemic extracellular
fluids in Ca2+ homeostasis.26,90,115 Indeed, the BLC membrane
expresses tight junction membrane proteins that are responsive to
metabolic demands such as chronic metabolic acidosis.116 More-
over, BLCs stain positive for osteoblast markers such as alkaline
phosphatase, (ALP) osteocalcin (OCN), and osteonectin, in
agreement with their osteogenic potential. BLCs are covered by
a thin layer of mesenchyme-derived cells called bone marrow
envelope (BME) cells that morphologically resemble BLCs and,
similar to BLCs, are considered osteoprogenitors.2
When the remodeling starts, BME cells and BLCs form a
protective structure known as the canopy, which separates
osteoclasts and osteoblasts from bone marrow117 (Fig. 3). At the
bone remodeling site, BLCs disconnect from the underlying
osteocytes through gap junction disruption, and digest fibrillary
collagen, the most abundant component in the bone extracellular
matrix (ECM), which is tightly packed in a helical structure to
provide mechanical stability and confer resistance against
proteolysis.118 To be degraded, fibrillary collagen usually requires
the activation of the cysteine protease cathepsin K and members
of the matrix metalloproteinase (MMP) family.119 While osteoclasts
express both of these enzymes types, which contribute differently
to bone resorption,120 BLCs do not express cathepsin K but can
efficiently remove nonmineralized fibrillary collagen (present
either on the quiescent bone surface or in resorption lacunae
after osteoclastic activity)113 mediated through their highly active
MMPs.121
Bone Research (2022)10:48
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S Bolamperti et al.
7
The generation of the canopy allows the formation of the bone
remodeling compartment (BRC), which is considered an essential
component of the BMU itself, as it provides a microenvironment
to link bone formation and resorption through local signaling.122
The BRC is generated in the proximity of the microvascula-
ture123 (Fig. 3). Although it is unclear how the BRC is formed,
several lines of evidence have suggested that the BRC favors a
direct connection between bone cells and sinusoids, which might
provide reservoirs of osteoprogenitors.122
In support of this hypothesis, an increased presence of capillaries
covering human cancellous bone has been found at remodeling sites
in an area within 50 µm from the canopy surface.124 This coverage
has also been observed on cortical bone remodeling sites.4
The tight link between BRCs and capillaries, which are coated
with pericytes, allows capillary-BRC canopy interactions to induce
BMU development at bone remodeling sites117 (Fig. 3). Pericytes,
first identified as cells covering microvessels/capillaries and
maintaining local homeostasis and angiogenesis, support the
mesenchymal niche and can be considered osteoprogenitors.125 It
has been hypothesized that pericytes first move from capillaries to
the canopy and then to the bone surface, where they differentiate
into mature osteoblasts during bone remodeling.126 It is also
plausible that the vascularization of the BRC favors the recruit-
ment of osteoprogenitors circulating in the peripheral blood.127
Recently, a specific capillary subtype that supports perivascular
osteoprogenitor differentiation by producing Nogging via Notch/
Dll 4 signaling has been termed type H vessel, which express both
CD31 and Endomucin at high levels. Type H vessels are
surrounded by abundant osteoprogenitors (Osterix-positive cells)
and are located both in the metaphysis, close to the growth plate,
and in the diaphysis (periosteum and endosteum).128,129 During
bone remodeling, the release of platelet-derived growth factor
type BB (PDGF-BB) from preosteoclasts induces an increase in
endothelial cell recruitment and assembly, with a subsequent rise
in the number of type H vessels. Simultaneously, PDGF-BB triggers
the recruitment of osteoprogenitors from vessels to the bone
surface.130 The formation of H vessels is supported by the
production of the slit homolog 3 (SLIT3) protein in osteoclasts,131
which also enhances H vessel branching.131–133
Type H vessels respond to the administration of intermittent
anabolic PTH (iPTH), which promotes the detachment of leptin
receptor-expressing (LepR+) cells, which are pericytes that can
differentiate into osteoprogenitors,134 from CD31hi/Edmhi vessels.
Pericyte migration contributes to the osteoprogenitor recruitment
to the BRC during PTH-induced bone remodeling.135 Notably,
anabolic responses to iPTH as well as sclerostin inhibitor
treatments, decline with time,136 as it is conceivable that the
precursor pool tends to be exhausted after major stimulation.
BLCs do not exclusively represent a final differentiation stage of
the osteogenic line; they can also be osteoblast precursors.137 A
study by Matic et al.137 led to the development of a model in
which osteoclasts initiate osteoprogenitor cell expansion (see
following section) by activating osteoprogenitor reservoirs located
in proximity to the eroded bone surface consisting of BLCs,
canopy cells and pericytes.120
Hence, BLCs favor the initiation of bone formation by supporting
the recruitment of bone-forming osteoblasts from the canopy
upon the release of osteoclastic factors supporting bone formation,
independently of bone resorption (see following section). The
osteoclast-canopy interface is therefore the physical site where the
coupling of bone resorption to bone formation occurs.117
OSTEOCLASTS
Resorbing osteoclasts: osteoclastogenesis and excavation of
resorption lacunae
Osteoclasts are responsible for bone resorption, a process that is
accomplished in a fairly short time relative to that required for
 The operational process of bone remodeling
S Bolamperti et al.
8
bone formation. Osteoclasts originate from the hematopoietic
monocyte-macrophage lineage, residing within the granulocyte-
macrophage colony-forming unit. Osteoclastogenesis starts in the
bone marrow upon the release of RANKL and macrophage colony-
stimulating factor (M-CSF) from osteocytes and vascular endothe-
lial cells close to the bone surface.138 It then extends toward the
bone surface through the layer of BLCs along an “osteogenesis
route”, likely guided by physical (i.e., collagen) and biochemical
(i.e., sphingosine-1-phosphate, S1P) signals.139 In vitro experi-
ments have shown that osteoclasts access bone surfaces by
directly displacing the cells present on the bone surface.140 TGF-
β1 is important for inducing cytoskeletal reorganization via the
p38 pathway in osteoblastic cells, which elongate to generate cell-
free areas.141 The inhibition of c-src and subsequent actin
organization in osteoclast precursors prevent osteoclasts from
migrating through the osteoblast layer.142 Metalloproteinase
(MMP) inhibitors prevent both osteoblast elongation and the
subsequent generation of cell-free areas143 and directly act on
osteoclasts by preventing their migration through the cell layer.141
However, in osteoclasts, MMP inhibitors did not affect actin
organization per sé,142 although they had been previously
demonstrated to prolong the podosome lifespan in these cells.144
MMP inhibitor administration fully prevented osteoclast recruit-
ment in the diaphysis core in vivo145 as well as osteoclast
migration through collagen in vitro.146 Among MMPs, MMP-14
and MMP-9 seem to play a major role in osteoclast migration.
MMP-14, also known as MT1-MMP, is expressed in all skeletal cells
but is more abundant in osteoclasts.147 MT1-MMP-knockout (KO)
mice usually die couple of months after birth, and display a severe
phenotype that includes delayed ossification, unclosed sutures
and unremoved cartilage.148 MMP-14 is highly expressed on
osteoclast podosomes,149 and it digests interstitial collagen as well
as other ECM molecules.150 MMP-9-KO mice showed reduced
osteoclast invasion into the core of developing bones,151 and
in vitro, osteoclasts lacking MMP9 did not migrate.152 However,
very recently, it has been demonstrated that osteoclasts lacking
either MMP9 or MMP14 alone (in this case conditionally deleted in
the myeloid population) did not show altered fusion and function,
or any defects in bone resorption. Only the deletion of both MMP9
and MMP14 generated a reduction in bone resorption areas and in
type I collagenolysis activity.153
The fusion of the earliest osteoclast precursors into multi-
nucleated, mature osteoclasts is a critical process in osteoclasto-
genesis, as the number of nuclei per cell determines osteoclast
aggressiveness.154 The presence of distinct giant hypernucleated
osteoclasts per sé in patients undergoing long-term treatment
with nitrogen bisphosphonates (NBPs), as discovered by Weinstein
et al.155 and confirmed by others,156 did not necessarily indicate
an increase in osteoclast aggressiveness. These osteoclasts indeed
showed decreased resorption competency and prolonged apop-
tosis characteristics. A recent study with transmission electron
microscopy demonstrated that NBPs first induce osteoclast
apoptosis and then the formation of newly differentiated
osteoclasts that can anchor the bone surface but cannot form a
clear zone or ruffled border.157 NPBs induce osteoclasts to acquire
a nonresorbing phenotype by inhibiting c-Src, which is essential
for cytoskeletal construction,158 and by stimulating dendritic cell-
specific transmembrane protein (DC-STAMP),159 which is neces-
sary for cell fusion.
Under physiological conditions, osteoclast fusion requires
immobility and heterogeneity between fusion partners; fusion
can only occur at the bone surface and must be completed for
successful resorption.139 As reviewed elsewhere,160 heterogeneity
is an integral feature of osteoclast fusion and may be related to
the capacity of fusion precursors to adapt to specific bone
microenvironments. Fusion also requires RANKL, which is highly
expressed by BLCs;161 osteoblasts in the growing skeleton; and
osteocyte arborizations that reach the bone surface in adults.51,52
sRANKL or mRANKL produced by osteogenic cells binds to RANK
on osteoclast precursors to activate intracellular signaling of tumor
necrosis factor receptor-associated factors (TRAFs), especially TRAF
2, 5, 6, which are adapter molecules that trigger NF-κB
activation.162 NF-κBs are transcription factors that were originally
discovered as regulators of B lymphocyte differentiation163 and
that play important roles in innate and adaptative immune
responses.164 Mice lacking both NF-κB 1 and 2 display, among
other features, severe osteopetrosis165 due to an accumulation of
osteoclast precursors.166 NF-κBs can activate canonical signaling
and noncanonical signaling pathways in osteoclasts. Canonical NF-
κB signaling activation is very fast, initiated within one hour of
RANKL binding, and leads to the induction of NFATc1 expres-
sion.167 NFATc1 is a transcription factor that plays a master role in
osteoclastogenesis168 by regulating the expression of osteoclast-
specific genes such as cathepsin K, DC-STAMP, and tartrate-
resistant acid phosphatase (TRAP) or that of osteoclast-associated
receptor (OSCAR) and other genes involved in OC resorptive
functions.169 The role of the Nf-κB noncanonical pathway in
osteoclastogenesis is controversial. Global deletion of single
molecules involved in the Nf-κB noncanonical pathway did not
affect osteoclast numbers in vivo,170 whereas conditional deletion
of TRAF3, an inhibitor of the noncanonical pathway, in osteoclast
precursors resulted in increased osteoclast formation and bone
resorption in mice.171
The discovery of a forward signaling induced by RANKL binding
to its receptor RANK on osteoclast precursors led to the
development of denosumab (DMAB), a human monoclonal
antibody that binds RANKL and inhibits osteoclastogenesis.
Recently, a second receptor for RANKL, the leucine-rich repeat-
containing G protein-coupled receptor LGR4, was discovered on
the osteoclast membrane. The extracellular domain of LGR4 binds
RANKL and inhibits NFATC1 expression, thus blocking osteoclas-
togenesis.172 Very recently, a variant of RANKL with changes in a
few amino acids in the RANK binding site was developed. This
variant still activates LGR4 signaling and inhibits osteoclastogen-
esis. Moreover, it has been observed that this variant acts as an
immunogen triggering the production of anti‐RANKL antibodies,
and its use might reduce the risks linked to the abrupt suspension
of denosumab use.173,70 The balance achieved among RANK,
RANKL, OPG, and LGR4 fine tunes bone resorption in the bone
remodeling process, and the pharmacological modulation of this
balance has critical therapeutic potential in osteoporosis.172
In addition to RANKL/RANK, LGR4 and M-CSF signaling174
guiding and balancing the early steps of osteoclastogenesis, other
factors act in concert to determine the osteoclastic mature cell
phenotype, including β3-integrin,175 NR4A1,176 immunoreceptor
tyrosine-based activation motif-containing proteins (ITAMs),
DNAX-activating protein and the Fcγ receptor.177 Additionally,
Toll-like receptors (TLRs) expressed on osteoclast progenitors178
promote differentiation toward the mature cell phenotype, as
observed in inflammatory osteolytic diseases.179
WNT proteins such as Wnt5A in the noncanonical pathway have
been outlined as key elements in osteoclastogenesis. WNT5a
produced by osteoblasts supports osteoclastogenesis by stimulat-
ing RANK expression in osteoclast precursors,180 promoting
osteoclast fusion,181 and supporting actin ring formation and
bone resorption via c-Src.182 Since WNT5a enhances osteogenic
pathway of Wnt/β-catenin activation in osteoblasts,183 it was
thought that WNT5a produced by osteoblasts might affect both
resorption and formation. However, recent studies have led to
modifications of this previous assumption as they demonstrated
that, after RANKL stimulation, osteoclast precursors also produce
and secrete a unique phosphorylated form of WNT5A. Further-
more, deletion of this phosphorylated form of WNT5a in mature
osteoclasts led to reduced bone formation in male mice.184 Hence,
it is speculated that in the BRC, osteoblast WNT5a first supports
osteoclastogenesis; then, as the osteoclast precursors differentiate,
Bone Research (2022)10:48

Osteoblast precursors
MIGRATION
TGF-β1
Wnt1
Osteoclast
MINERALIZATION
ECM Wnt10b
Fig. 4 Released extracellular matrix (ECM) factors. Two major factors are released by the ECM upon bone resorption and cooperate to regulate
bone remodeling and BMU activity: TGF-β1 and IGF-1. After release from the matrix, active TGF-β1 acts both on osteoblasts and osteoclasts to
induce osteoblast precursor migration to the site of resorption and osteoclast production of Wnt1, which promotes osteoblast recruitment
and/or differentiation at sites of bone resorption, and Wint10b, which promotes matrix mineralization. IGF-1 supports the recruitment of
osteoblast progenitors and promotes osteoblast differentiation and matrix mineralization by inducing the transcription of osteogenesis‐
related genes such as DMP1, PHEX, SOST, BMP2, RUNX2, OPN, and OCN
WNT5a expression increases, leading to the subsequent enhance-
ment of osteoclast fusion and activity. In later stages, WNT5a
production by mature osteoclasts fosters osteoblastogenesis
because WNT5a cooperates with canonical Wnt signaling. The
subsequent expansion of the osteoblast pool might therefore
fulfill the resorption cavity at the proper time and space,
suggesting that Wnt5a is a clastokine that maintains
osteoclast–osteoblast coupling in physiological bone remodeling.
The cytoskeletal and membrane organization of osteoclasts
characterizes their differentiation state. Mature osteoclasts are
polarized cells with an apical membrane facing bone and an
opposite basolateral membrane facing plasma. Both apical and
basolateral membrane domains are essential structures for
resorbing osteoclasts.185 Critical functional features of the apical
membrane domain are the sealing zone and the ruffled border.
The sealing zone establishes contact with the bone surface and
delimits the resorption lacunae (i.e., Howship’s lacunae). The
ruffled border membrane allows large vesicle transport, the
delivery of hydrochloric acid (by vacuolar-type H + ATPase186)
and chloride ion channel-7 (CLC-7),187 and the release of several
lysosomal proteases, such as cathepsin K, TRAP, and MMPs.188
After endocytosis, the products derived from matrix degradation
are transported through the cell within vesicles for further
intracellular degradation and exocytosed on the opposite site
through the secretory domain at the basolateral membrane.189
Thus, the osteoclasts never lose their tight attachment to the bone
surface when resorption occurs. The interaction with the matrix is
mediated by the integrin complex α5β3, which recognizes RGD
motifs in proteins such as fibronectin,190 osteopontin (OPN)191 and
bone sialoprotein.192 By binding to RGD motifs, the α5β3 complex
is activated and clusters at the ruffled border,193 thus allowing the
activation of focal adhesion complexes and the formation of the
sealing zone during resorption.194 It has been hypothesized that
integrin β3 contains two motifs with different affinities (high and
low) for Ca2+ concentrations. A normal Ca2+ concentration in BEF
maintains low-affinity domain activation, which does not maintain
a strong attachment between β3 and RGD. When Ca2+
concentrations diminish, such as when osteocytes are injured,
the high affinity site is the only one activated; this motif induces
αvβ3 clustering, which allows osteoclasts to strongly adhere to the
matrix and initiate bone resorption.195
The overall fate of osteoclasts (i.e., recruitment, differentiation,
fusion and apoptosis, see below), is tightly controlled by the
process coordinating bone remodeling, in which osteoclasts are
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The operational process of bone remodeling
S Bolamperti et al.
9
TGF-β1
IGF-1
Wnt1
DIFFERENTIATION BMP2, Wnt10b
RUNX2,
OPN, DMP1,
IGF-1 PHEX, SOST
TGF-β1 receptor
IGF-1 receptor
Osteoblasts
Wnt receptor
Resorption lacuna
also providers of multiple coupling signals196 for osteoblast
formation.
Resorbing osteoclasts: the release of bone matrix factors
The bone ECM resorbed by osteoclasts contains inorganic and
organic compounds. A growing body of evidence in ECM biology
suggests that several organic proteins of the matrix regulate the
quantum of BMU activity. While the only inorganic components
of the ECM are apatite and trace elements, proteomic analysis
have led to the identification of more than 100 organic ECM
proteins,197 with collagen type I (Coll-1) and noncollagenous
proteins (NCPs) being the major constituents. NCPs can bind
growth factors, membrane receptors, and adhesion molecules,
forming an intrinsic biochemical signaling network within each
BMU. NCPs comprise several Gla proteins, including bone-Gla
protein (i.e., Osteocalcin, OCN), matrix-Gla protein (MGP),
protein-S, Gla-rich protein (GRP), periostin, and periostin-like
factor (PLF), as reviewed elsewhere.198 In addition to OCN, whose
hormonal effects were previously established in animal mod-
els,199 Gla proteins exhibit several functions supporting bone
metabolism through γ-carboxylation-dependent and γ-carbox-
ylation-independent mechanisms, ranging from the regulation
of cell adhesion and activity to the modulation of calcium
concentrations in the extracellular space. Gla proteins act in
concert with glycoproteins such as osteonectin, thrombospon-
dins and R-spondins, sialoprotein and matrix extracellular
phosphoglycoprotein (MEPE) and DMP1 to control mineraliza-
tion, synergistically with FGF23 and PHEX produced by
osteocytes (see Lin et al.200 for review).
Two major factors released by the ECM influence bone
remodeling and BMU activity: transforming growth factor-β
(TGF-β) and insulin growth factor-1 (IGF-1). Together, TGF-β and
IGF-1 influence the recruitment and differentiation of osteoblast
lineage cells (Fig. 4), favor bone matrix mineralization, and
regulate osteoclast activity. For these reasons, they are acknowl-
edged as “coupling factors”.
TGF-β. TGF-β1 is one of the most abundant cytokines in the
bone matrix.201 TGF-β1, which is a member of the TGF superfamily
that includes bone morphogenetic proteins (BMPs), binds to
specific TGF receptors (TGFR1-2) in osteoblasts, where it activates
the synthesis of collagen and the expression of RUNX2, a master
transcription factor regulating osteoblast proliferation and differ-
entiation. TGF-β1 release and activation, resulting from the activity
 The operational process of bone remodeling
S Bolamperti et al.
10
of metalloproteinases and interaction with integrins, are pre-
requisites for the exertion of TGF-β1 effects on bone cells. This
occurs during bone resorption since osteoclasts lower the pH in
resorption lacunae and secrete cathepsin K, thus releasing and
activating TGF-β1.202 When osteoclasts actively resorb bone, TGF-
β1 is released in the lacunae, thus playing a crucial role in the
balance between bone formation and resorption. First, active TGF-
β1 induces osteoblast precursor migration to the site of
resorption203 without affecting the differentiation of cells.204 Since
TGF-β1 alone is not able to promote osteoblast differentiation,205
cooperative pathways need to be activated to foster an
osteogenic environment at resorption surfaces within the BRC. A
cooperation pathway is provided by the osteoclast itself. In
addition to activating and releasing TGF-β1 from the bone matrix,
osteoclasts express specific receptors for TGF-β1. Osteoclastic TGF-
β1 receptor signaling stimulates the production of Wnt1, which
promotes osteoblast recruitment and/or differentiation at sites of
bone resorption. Since impaired TGF-β1 receptor signaling in
osteoclasts has detrimental effects on bone mass in mouse
models, it is likely that the release of Wnt1 constitutes a coupling
pathway.206 Moreover, TGF-β1 favors RANKL production in
osteoblast precursors by inducing TRAF3 degradation. The
expression of TRAF3, which is an inhibitor of noncanonical Nf-κB
signaling, in osteoblast precursors favors the stabilization of
β-catenin and, therefore, their differentiation into mature osteo-
blasts. In the absence of TRAF3 expression, Nf-κB noncanonical
signaling is activated with an increase in RANKL production and
therefore is associated with osteoclastogenesis.207 With matrix
resorption progression, the concentration of TGF-β1 increases, and
TGF-β1 activates a negative feedback loop in osteoclastogenesis.
High TGF-β1 expression inhibits the migration of osteoclast
precursors208 by suppressing RANKL expression and by stimulat-
ing OPG production in osteoblasts.209 A reduced RANKL/OPG ratio
indeed results in reduced osteoclastogenesis. In the absence of
osteoclast survival factors such as RANKL, TGF-β1 leads to
osteoclast apoptosis via the upregulation of Bim expression.210
This proapoptotic effect of TGF-β1 on osteoclasts is consistent
with the crucial role it plays in the reversal of bone resorption to
bone formation. Indeed, TGF-β1 release enhances Wnt10b
expression and secretion to stimulate osteoblast-directed miner-
alization. TGF-β1 therefore has a dual effect on osteoblasts by
directly recruiting their progenitors to the bone surface and by
indirectly promoting matrix mineralization through osteoclast-
derived Wnt10b.211
IGF1. IGF-1 is the most abundant growth factor deposited in
the bone ECM.212 Osteocytes in particular secrete large
amounts of IGF1, which is incorporated in the bone matrix.213
Since IGF-1 is produced under loading, it might contribute to
the mechanotransduction process.214 Whether released by cell
secretion or bone ECM degradation, IGF1 is a local autocrine/
paracrine regulator of bone remodeling and does not con-
tribute to the circulating IGF-I pool. IGF-1 acts primarily as a
migratory signal and as a differentiation factor for osteoblast
precursors, rather than as a proliferation factor. IGF-1 recruits
progenitor cells of the osteoblastic lineage215 and promotes
osteoblast differentiation and matrix mineralization by upregu-
lating the osteogenesis‐related genes DMP1, PHEX, SOST, BMP2,
RUNX2, OPN, and OCN.216 Moreover, IGF-1 released from the
bone matrix during bone resorption establishes an osteogenic
microenvironment by activating mechanistic target of rapamy-
cin (mTOR) signaling,217 which is critical for cellular energy
metabolism and cell migration.218 Recent studies have con-
firmed that IGF-1-dependent activation of mTOR induces
human dental pulp stem cell (DPSC) differentiation toward an
osteoblastic phenotype.219
Since TGF-β1 and IGF1 do not remain accessible long enough
to affect the complete refilling of the resorption cavity,220 their
action might be limited to the early phases of remodeling,
when osteoclasts are still actively resorbing the bone matrix.
ANABOLIC OSTEOCLASTS: A BRIDGE FROM BONE RESORPTION
TO FORMATION
The resorption of the bone ECM is necessary, but not sufficient, to
induce the stimulus provided by osteoclasts to osteoblasts during
remodeling. Currently, available evidence indicates that osteo-
clasts can support bone formation independent of their resorption
activity. The paradoxical concept of “anabolic osteoclasts” was
articulated after the original observation that human osteoblasts
exposed to conditioned media from human osteoclasts increased
bone nodule formation.221 The finding that the transplantation of
nonfunctional osteoclasts in irradiated skeletally mature 3-month-
old wild-type mice induced a high trabecular bone volume,
increased bone strength, and an increased bone formation rate in
trabecular bone222 supports this concept. The anabolic role of
osteoclasts can be observed in both humans and mice with
osteoclast-rich osteopetrosis (OPT), a disease resulting from
mutations of either the V-ATPase subunit or the ClC-7 chloride
channel that abrogate the osteoclastic acidification process, which
is essential for resorption activity. Under these conditions, no
factors stored in bone were released from the unresorbed bone
matrix, but the long-surviving osteoclasts, fused into large
abnormal multinucleated cells, provided an osteogenic milieu.223
In fact, in models of osteoclast-rich OPT, bone formation is
maintained or enhanced despite the impairment of resorption
activity,224 thus providing evidence that nonresorbing osteoclasts
promote/maintain bone formation. Findings following the phar-
macologic blockage of cathepsin K in osteoclasts (Odanacatib) are
in line with this view. In patients treated with Odanacatib,
although osteoclast bone resorption was impaired, the osteoclast
number was increased to be more than twofold higher than that
after placebo treatment by month 60, and bone formation, after a
transient reduction, was maintained.225
In contrast to “osteoclast-rich” OPT, active bone formation is
lacking in “osteoclast-poor” OPT (due to TNFRSF11A mutations226)
or dysosteosclerosis (DSS) (an OPT of unknown etiology227).
Indeed, bone histomorphometric analysis showed no linear single
or double tetracycline labels under these conditions.
Therefore, these “anabolic osteoclasts” might produce a pool of
signals that activate bone formation and that can be a) released
into the BRC, b) shuttled to osteoblasts, and c) transferred by
receptor binding.
Information released into the BRC
Several osteoclast-derived factors released into the BRC have been
identified and validated in genetically modified mouse models.
These factors include cardiotrophin-1 (CT-1),228 S1P,229 Wnt10B,
BMP6,230 collagen triple helix repeat-containing 1 (CTHRC1),231
complement factor 3a (C3a),232 and leukemia inhibitory factor
(LIF).233 However, not all of these identified factors are responsible
for coupling in bone remodeling, nor are they produced only by
osteoclasts due to macrophage contamination in vitro and the
presence in vivo of immunocompetent cells within the remodel-
ing compartment. Whether these factors exhibit similar effects in
humans remains unclear; however, a recent study shed some light
on this controversy. Bone biopsy samples taken from postmeno-
pausal women treated with DMAB were analyzed by RNA
sequencing, and the results were compared to those obtained
from bone biopsy samples of untreated subjects. The comparison
revealed potential osteoclast-derived coupling factors in humans:
LIF, CREG2, CST3, CCBE1, and dipeptidyl peptidase-4 (DPP4), a
highly conserved cell surface peptidase. The reduction in
osteoclast-derived DPP4 in the DMAB-treated group was asso-
ciated with a significant increase in glucagon-like peptide-1
(GLP)−1 in the serum compared to that in the placebo-treated
Bone Research (2022)10:48

Monocyte/Macrophage
RANK
RANKL
Forward signaling RANKL
OC
OB
Apoptotic OC EphB2 EphB4 R
TO
m
RvCs
RANKL
vesicles
Reverse signaling
Apoptotic
bodies
Fig. 5 Anabolic osteoclasts. Osteoblasts stimulate osteoclastogen-
esis by producing RANKL as a forward signal. However, osteoclasts
can act as anabolic cells by generating positive reverse signaling in
osteoblasts. Both resorbing and apoptotic osteoclasts can release
extracellular vesicles (EVs) that contain RANK. Once discharged from
EVs, RANK binds RANKL clusters on the osteoblast membrane and
activates osteogenesis via the mTOR pathway. In addition,
EphrinB2 signaling from osteoclasts to EphB4 on osteoblasts/bone
lining cells (BLCs)/reversal cells favors osteogenic differentiation
group, suggesting that DPP4, in addition to its function as a
coupling factor, might constitute a potential link between bone
remodeling and energy metabolism.234
Mature human osteoclasts also secrete SLIT3, a chemorepellent
originally identified as a regulator of axon crossing in the brain.
Osteoclast-derived SLIT3 stimulates the migration and prolifera-
tion of osteoblast lineage cells via the activation of β-catenin and
suppresses osteoclastogenesis in an autocrine manner.131 SLIT3
contributes to the initial establishment of the osteogenic
environment after resorption and meets the requirements to be
considered a coupling factor, as i) SLIT3 production increases
during osteoclast differentiation and ii) SLIT3 is necessary for
osteoblast precursor migration.235 Considering that bone greatly
contributes to SLIT3 levels in plasma, SLIT3 might be considered a
novel biomarker of bone turnover and a candidate for the
treatment of osteoporosis because it plays active roles in both
bone resorption and formation through its opposite effects.
Information shuttled to osteoblasts
The coupling of osteoclasts to osteoblasts and related functions
involve not only the secretion of factors in the BRC but also the
production of specific EVs, released by bone cells. EVs have
emerged as important intercellular regulators since they function
as messengers from osteoclasts to osteoblasts, and vice versa.236
EVs exhibit an evolutionary advantage over the paracrine
pathway activated by released factors because they can protect
their message (mRNAs and proteins, including cytokines, etc.)
from degradation and might therefore play critical roles in fine-
tuning bone remodeling.
Osteogenic cells trigger hematopoietic precursor differentiation
into osteoclasts by secreting RANKL. In addition, through a less-
characterized mechanism, osteoclasts stimulate osteoblasts using
the same RANKL–RANK pathway acting in reverse mode; that is,
RANKL acts as the receptor. In fact, osteoclasts release EVs
(between 25 and 120 nm in diameter) that carry RANK. These
vesicles were initially thought to be negative paracrine regulators
of osteoclastogenesis.237 However, a recent study demonstrated
Bone Research (2022)10:48
The operational process of bone remodeling
S Bolamperti et al.
11
that vesicular RANK binds to RANKL on the osteoblast surface (and
possibly on osteocytes) and triggers the mTOR-dependent
stimulation of Runx2238 (Fig. 5). This finding suggests a previously
unknown scenario in which actively resorbing osteoclasts promote
the differentiation of neighboring osteoblast precursors into
mature bone-forming cells. The physiological importance of the
messages shuttled by EVs has been confirmed by the observation
that osteoblasts do not efficiently deposit bone ECM when RANKL
reverse signaling is suppressed.238
The discovery of the reverse signaling induced by RANK on
osteoclasts binding to RANKL on osteoblasts indicates new
pharmacological possibilities. Interestingly, the administration
of a specific peptide, W9, to prevent RANKL-RANK signaling has
been shown to exert a bone anabolic effect in vivo by inducing
uncoupling.239 Therefore, DMAB may activate RANKL reverse
signaling,240 explaining the continuous increase in bone
mineral density (BMD) observed at the 10-year follow-up of
DMAB-treated individuals.241 However, experimental evidence
supporting the hypothesis of the bimodal effects of DMAB on
RANKL-RANK signaling during bone remodeling is lacking
because remodeling-based bone formation is severely sup-
pressed in the femur of DMAB-treated subjects.242 It is more
likely that DMAB blocks RANKL expressed on bone marrow
mesenchymal stem cells (BMSCs),243 through which it nega-
tively regulates osteogenic differentiation.244 According to a
hypothesis suggested by Wang et al.,243 the DMAB-related
abrogation of RANKL-induced inhibition of osteoblast precur-
sors might underlie the activation of modeling-based bone
formation242 and sustain the increase in BMD over time, as
previously observed.241
RANKL monomers expressed on osteoblasts are not activated
by RANK238 or by OPG, as indicated by the deletion of RANK or
OPG failing to reduce bone formation.245 In contrast, the
multimeric assembly of RANKL can activate reverse signaling. In
fact, OP3-4, a RANKL-binding peptide that induces the clustering
of RANKL on the cell membrane, induces the activation of RANKL
reverse signaling.246 RANKL clustering on early osteoblasts may
therefore be required for the activation of RANKL reverse signaling
in resorption lacunae, similar to the effect of shuttling RANK in
osteoclast EVs during bone remodeling (Fig. 5) or that of an anti-
RANKL antibody-containing leucine zippers, which induce trimer
formation.238
Some of the exosomes released by osteoclasts at the end of
the two-week cell life span contribute to signal transfer during
remodeling. In fact, apoptotic osteoclasts produce large amounts
of apoptotic bodies. Apoptotic bodies are subcellular membrane-
bound EVs containing fragments of nucleus, endoplasmic
reticulum and mitochondria that assemble in a random manner
and are involved in intercellular crosstalk.247 Osteoclast-derived
apoptotic bodies contain osteoclast differentiation factors, such
as RANK and RANKL (Fig. 5). In contrast, osteoblast-derived
apoptotic bodies contain specific osteogenic factors, such as
BMPs, OPN, OCN and bone ALP (BALP). By mapping the whole-
proteome signatures of osteoclast-derived apoptotic bodies, the
apoptotic bodies content was confirmed to be consistent with
that of their parent cells in terms of both proteome signatures
and biological functions.248 During remodeling, osteoclast-
derived apoptotic bodies containing miR-214-3p are delivered
to osteoblasts and serve as intercellular messengers that inhibit
osteoblast differentiation and subsequent bone formation.249
Recently, the roles of osteoclast-derived apoptotic bodies have
been more precisely characterized, and their effects have been
found to be determined by the differentiation state of parental
cells.250 For example, apoptotic bodies derived from mono-
nuclear preosteoclasts undergoing apoptosis deliver PDGF-BB to
recipient endothelial progenitor cells (EPCs), and since PDGF-BB
is a proangiogenic factor,130 preosteoclast derived apoptotic
bodies promote angiogenesis. However, apoptotic bodies
 The operational process of bone remodeling
S Bolamperti et al.
12
derived from multinucleated mature osteoclasts promote osteo-
genesis via RANKL reverse signaling.
Therefore, in addition to well-studied osteoclast–osteoblast
coupling, the involvement of osteoclast-derived apoptotic
bodies promotes the transition from bone resorption to
formation during bone remodeling. Although the mechanism
by which apoptotic bodies delivery is controlled within each
BMU is unclear, mature osteoclasts derived apoptotic bodies
further connect the disappearance of osteoclasts from resorption
lacunae with the incoming osteogenic process involving
osteoblast recruitment, migration and differentiation, contribut-
ing to the reversal of cellular activity in reverting from bone
resorption to bone formation.
Information transferred by receptor binding
The communication route between osteoclasts and osteoblasts
involves a bidirectional signaling pathway mediated by EphrinB2‐
EphB4 that links the suppression of osteoclastogenesis to
osteogenesis within each BMU.251 EphrinBs are transmembrane
proteins with cytoplasmic domains that are preferential ligands for
the tyrosine kinase receptor EphB. EphrinB2 expression in
osteoclasts is associated with RANKL-induced osteoclast differ-
entiation, whereas EphB4 is constitutively expressed in osteo-
blasts. Signaling from EphrinB2 in mature osteoclasts to EphB4 in
osteoblast precursors stimulates osteogenic differentiation (Fig. 5),
whereas signaling from EphB4 in osteoblasts to EphB2 in
osteoclasts inhibits the differentiation of osteoclast precursors.
EphrinB2–EphB4 bidirectional signaling must function locally and
requires direct cell‐cell contact since both receptors are anchored
to the cell membrane, as shown with goldfish scale model of bone
remodeling.252 In humans, the role of EphrinB2 and EphB4 in bone
remodeling has been debated because osteoclasts actively
resorbing bone, and osteoblasts forming bone do not directly
get in contact in the BRC. As a result the cell–cell interaction
mediated by EphrinB2 and EphB4 might be limited to the
precursor stage, when osteoclastogenesis and osteoblastogenesis
are simultaneously regulated, or (as suggested previously253) to
the mature osteoclast stage, in which direct contact is realized
with the BLCs surrounding the canopy. Under PTH1R activation,
EphrinB2 expressed on osteoclasts has been suggested to affect
EphrinB4 on the osteoblast/BLC surface to induce osteoblast
commitment and promote osteoblast differentiation.254
INTERMEDIATE CELL PHENOTYPES: OSTEOMORPHS AND
REVERSAL CELLS TUNING AND COUPLING BONE REMODELING
The orderly genesis and apoptosis of both osteoclasts and
osteoblasts are essential for physiological bone homeostasis
during bone remodeling. Possible intermediate phenotypes may
be appropriate targets for coupling factors by providing a temporal
connection between osteoclasts and osteoblasts. In the traditional
view, osteoclasts were thought to live for two weeks11 before
undergoing apoptosis; it was therefore assumed that during bone
remodeling, the osteoclast resorption phase within a single BMU
was temporally limited by the osteoclast life span, ending with the
completion of its bone-resorbing activity. However, the observa-
tion that osteoclasts can survive longer by fusing with circulating
monocytes with access to BRC255 has challenged this view. While
osteoclast apoptosis is a rare event requiring high energy
expenditure for the removal of the apoptotic debris, osteoclast
disassembly into smaller unresorbing cells, which can revert to
functional osteoclasts when needed, is a more efficient process.
Two observations support the latter view: first, primary osteoclasts
have been identified at the cutting cone and secondary osteoclasts
have been shown to establish a functional link with reversal cells
(Figs. 1 and 3) as focal bone remodeling progresses within a single
BRC,256 and second, osteoclasts have recently been shown to be
recycled into osteomorphs. Osteomorphs have been described as
highly motile osteoclast daughter cells that remain in the adjacent
bone marrow and retain the ability to fuse back into functional
osteoclasts.257
The discovery of a transient cell stage of osteoclasts (i.e., the
osteomorph) that can undergo phenotype reversion, analogous to
what occurs in osteoblasts (i.e., BLCs), implies the presence of two
reacting components of the BMU that can rapidly adjust the
operational process of bone remodeling. This implication is
clinically relevant to osteoporosis therapy. For example, BLCs
can react to PTH exposure by quickly reverting to osteoblasts and
thus expand the osteoblastic pool258 to fill resorption lacunae,
similar to process in the “anabolic window” after teriparatide and
abaloparatide administration.259 Similarly, exposure to RANKL
might allow osteomorphs to rapidly fuse and recycle back into
functional osteoclasts. Since RANKL inhibition by DMAB results in
osteomorph accumulation,257 the reconstitution of RANKL signal-
ing, as occurs upon DMAB discontinuation, can activate massive
bone resorption over a very short time scale, leading to rebound
fractures.260 This rebound effect is not fully inhibited by bispho-
sphonate therapy,261 probably because osteomorphs first accu-
mulate at bone sites and are thus not accessible to potent
bisphosphonate treatment. After osteoclasts are no longer active
and disappear, the Howship’s lacunae remain covered with
undigested nonmineralized collagen matrix. Mononucleated cells
colonize these eroded surfaces (ESs), covering 80% them.262 These
cells, called reversal cells,263 lack specific cell identification
markers. The available literature has identified the cells appearing
at the resorption lacunae as osteoclasts264 or, more recently, as
osteoblasts.265 Indeed, the reversal cells express osteoblast lineage
cell markers such as Runx2, ALP, and Col3, but not TRAcP or
CatK.265 Indeed, reversal cells express osteoblast markers such as
Runx2, ALP, and Col3 but not TRAcP or CatK.265 Abdelgawad
et al.266 showed that the immunoreactivity of these cells for
osteoclast-derived TRAcP is attributable to TRAcP taken up by
osteoblast lines from neighboring osteoclasts and does not
represent the acquisition of an osteoclastic phenotype. Moreover,
Abdelgawad et al.266 observed that early reversal cells, located in
close proximity to resorbing osteoclasts and forming direct cellular
contacts with neighboring osteoclasts through short cytoplasmic
extensions, are osteoblast progenitors with the capacity to digest
fibrillar collagen remnants, similar to BLCs.113 In particular, reversal
cells have been shown to be distributed among osteoclasts in the
resorption surface far from the pockets of osteoclasts at the
cutting cone tip. New resorption events might occur after the
primary excavation of the resorption cavity/canal when reversal
cells have colonized ES.256 In contrast, osteoclasts have never
been observed at the bone formation wall, suggesting that bone
resorption continues until bone formation takes place. Lassen
et al.’s256 findings indicate that a pure reversal period, as
commonly depicted following a halt in resorption,8 does not take
place. In contrast, it is conceivable that the expanded pool of
reversal cells, initially interacting with bystander osteoclasts in the
same time and space, gradually switch off further resorption and
form an osteogenic environment, activating bone formation later,
when osteoclasts are completely absent.
The dynamic events described here support the view that the
operational process of bone remodeling is a continuous physiological
entity. All events, from the formation of the bone remodeling
compartment with canopy cells to the colonization of a reversal zone
by reversal cells in the early period of bone remodeling, generate the
environment necessary for the recruitment and expansion of the
osteoblast pool and subsequent refilling of resorption lacunae (Fig. 3).
THE OSTEOBLASTIC CELLS POOL: RECRUITMENT, EXPANSION
AND OSTEOCYTOGENESIS
Osteoblasts are cuboidal-shaped cells on bone surfaces. Osteo-
blasts have traditionally been considered the cell responsible for
Bone Research (2022)10:48

the production of the organic components of the bone matrix and
its consequent mineralization because they synthetize and secrete
matrix components such as collagen type I, OCN, osteonectin,
bone sialoprotein, OPN, proteoglycans, and ALP.267 In vivo, a set of
osteoblasts, all with similar morphologies, lay down osteoid on
active bone-forming surfaces.268 The dense endoplasmic reticu-
lum of these cells indicates high synthetic activity, and the
extensive contacts among these osteoblasts, osteocytes and BLCs
indicate their extensive intercellular connections.269
Marotti et al.270 hypothesized that in human bone physiology,
osteoblasts can participate in two different types of osteogenesis:
static and dynamic osteogenesis. Static osteogenesis occurs in the
mesenchymal tissue to initiate intramembranous ossification to
generate primary trabecula during growth. This osteogenesis type
is thus independent of the loading environment and will be
subject to later osteoclast resorption. Static osteogenesis involves
stationary osteoblasts, which are pluristratified and polarized in all
directions. These osteoblasts differentiate into osteocytes in the
exact same site where they had been originated from the
mesenchymal precursors.271 Dynamic osteogenesis takes place to
thicken the primitive trabeculae generated in the context of static
osteogenesis, and it is therefore the type of osteogenesis activated
in response to metabolic and mechanical demand. The osteo-
blasts participating in dynamic osteogenesis are arranged in an
epithelial-like manner within a single cell layer. They all are
polarized in the same direction, and they maintain contacts with
osteocytic dendrites as they move from the mineralization front
toward the vascular surface.271 In the adult skeleton, physiological
bone remodeling involves only dynamic osteogenesis, which refill
resorption lacunae.
Osteoblastic cells contribute not only to osteogenesis but also
to osteoclastogenesis by producing RANKL. The same osteo-
genic cells may accomplish this dual function at different stages
of maturation. Immature osteoblasts respond to bone regulatory
factors in a pro-osteoclastogenic manner, but during the
maturation process, they lose RANKL expression and do not
support osteoclastogenesis.272 Moreover, as cells mature, they
secrete more ECM and exert local inhibitory control over
osteoclastogenesis by releasing the osteoclastogenic inhibitor
OPG.77 Osteoblasts are considered the major sources of OPG in
cancellous bone, acting in concert with osteocytes and other
cells to modulate the RANKL/RANK ratio and reduce resorption
by osteoclasts. This inhibitory activity is mandatory for the
correct shift from bone resorption to bone formation and
protects newly formed bone against untimely resorption.77
Cawley’s study stressed the concept “that the local concentration
of OPG near the cell surface is a key factor in its ability to block the
activity of RANKL”.
The osteoblast lineage derives from the differentiation of
mesenchymal cells in the stromal compartment of bone marrow
(BM). The bone marrow stroma contains self-renewing, multi-
potent progenitors that can give rise to osteoblasts, thus ensuring
a reservoir of bone-forming cells for bone growth, modeling and
remodeling.273 Anatomically, the BM stroma is part of the skeleton
and is present in all bones except auditory ossicles, which
therefore do not undergo remodeling.274 The definition of skeletal
stem cells and their physiological significance and terminology
associated with them have been debated.273–275 A self-renewing
stromal progenitor, originally referred to as an “osteogenic” or
“stromal”/”stem” cell (BMSC), corresponds to a specific type of
perivascular cell in mammalian bone marrow. The characterization
of pericytes as multipotent progenitors is evolving. Nevertheless,
Sacchetti et al.276 pointed out that only BM vessel-residing cells
defined as pericytes have the ability to differentiate into
osteoblasts during physiological bone remodeling. Since tissue-
specific mesodermal progenitors show tissue-specific commit-
ments in vivo, the native skeletogenic potential is thus intrinsic to
the progenitor/stem cell set found in the skeleton.277
Bone Research (2022)10:48
The operational process of bone remodeling
S Bolamperti et al.
13
Upon the initiation of bone remodeling, as previously discussed,
the formation of BRCs by BLCs/canopy cells establishes a direct
connection with the vascular sinus closely covering bone-forming
surfaces, allowing pericyte detachment and differentiation toward
the osteogenic phenotype.
Cells with true osteogenic potential can also circulate.278 Cells
with multiple potential differentiation routes similar to those of
BMSCs can enter the circulation, and although the mechanism by
which they enter the circulation is unclear, they circulate in
physiologically significant numbers, correlate with markers of
bone formation and increase during bone growth.127 Character-
ization of these cells deserves further study, and circulating
skeletal progenitors might form an additional pool of osteoblast
precursors for bone remodeling and/or tissue repair. In fact,
fractures result in the mobilization of mesenchymal osteogenic
progenitors into the circulation to promote fracture healing, even
in very old patients.279 Although circulating osteoprogenitor fate
and function are determined by the homing of these cells to focal
sites and their exposure to microenvironment signaling,280 their
presence per sé in the blood has important implications for
regenerative medicine.281
The average lifespan of active osteoblasts is 3 months.4 When
bone matrix formation ends and the subsequent matrix miner-
alization process completes the primary phase, mature osteoblasts
undergo three possible fates. Between 60% and 80% of
osteoblasts at resorption lacunae die via apoptosis.282 Cells at all
differentiation stages undergo apoptosis, which continues
throughout the osteoblast life span and is regulated by Wnt
signaling, PTH and mechanical stimulation.61 Osteoblast apoptosis
is key to regulating the extent and duration of bone formation and
is the target of catabolic and anabolic regulatory factors affecting
bone mass. Apoptosis exerts a significant effect on the number of
osteoblasts producing bone matrix at a bone formation site.283
The balance between the expression of proapoptotic factors Bim
and Bak and the prosurvival factor Bcl determines the osteoblast
life span and, therefore, bone volume and strength.284 Estrogens
and androgens also exert effects on bone cell life span. In
particular, they exert antiapoptotic effects on osteoblasts and
osteocytes and proapoptotic effects on osteoclasts. Interestingly,
estrogen receptors or the androgen receptor can transmit
antiapoptotic signals with similar efficiency, regardless of whether
the ligand is an estrogen or an androgen.285 p53, a potent tumor
suppressor, is also critically involved in determining osteoblast life
span, as it induces apoptosis, inhibits osteoblast proliferation and
stimulates osteoblast differentiation through the Akt–FoxOs
pathway286 to regulate bone remodeling.287
The remaining osteoblasts (those on the bone trabecular
endosteal and endocortical surfaces) might differentiate into BLCs,
which are characterized by lower biosynthetic activity and a flat
morphology, or they might remain embedded in the matrix, undergo
the active process of osteocytogenesis,288 and become osteocytes
(5%–20% of mature osteoblasts). Osteocytogenesis requires proteo-
lytic activity to deepen the osteoid. Collagenase-resistant type I
collagen in the bone matrix increased the osteocyte apoptosis
rate.289 Additionally, the deletion of the metalloproteinase MT-MMP1
generated an altered osteocytic network in mice.290 Osteocytogen-
esis requires cytoskeleton plasticity to form dendrites (dendrogen-
esis). Although the entire dendrogenesis process has not yet been
elucidated, one factor known to be necessary in the dendrogenesis
of osteoid osteocytes (the late osteoblast/early osteocyte that
undergoes the transition to mature osteocytes288) is E11/gp38 or
podoplanin (Pdpn), a transmembrane glycoprotein. The conditional
deletion of PDNP in mice generated a decreased number and length
of osteocyte processes.291 Recently, it has been shown that SP7
(Osterix) is also necessary to support dendrogenesis because it
regulates Osteocrine.292
With the progression of osteocytogenesis, osteoid osteocytes
start expressing specific cytoskeletal markers (fimbrin and destrin)
 The operational process of bone remodeling
S Bolamperti et al.
14
and factors for mineralizing the surrounding matrix (Phex and
MEPE) until they become mineralizing osteocytes (expressing
Dmp1). The cell terminally differentiate into mature osteocytes
producing SOST and FGF23.288
The differentiation of osteoblasts into osteocytes at the end of
the bone remodeling process is essential to reestablish mechan-
ical competence. Therefore, 9.1 million osteocytes are replaced
daily during bone remodeling.16 Hence, osteocytes can be
considered dynamic cells capable of reconstituted mechanical
sensitivity after replacement293 as well as the inhibitory control
over osteoclastogenesis via the local production of OPG.77
CONCLUSIONS: BONE REMODELING AS A CONTINUOUS FLOW
OF INFORMATION AND CONNECTED EVENTS
Current evidence on bone remodeling has indicated the complex-
ity of this process by showing how events and effectors are
connected and coordinated to ensure the removal of old bone
and maintain bone mechanical competence. Recent findings have
shown that bone remodeling is a physiological process consisting
of continuous operations with fast reactive components that
confer survival advantage. After BRC formation, local coordinating
factors and events, acting in concert in time and space and
responding to metabolic and mechanical regulators, affect the
final quantum of BMU activity. This complete picture overwrites
the classical view of bone remodeling organized in discrete stages.
The current knowledge indicates that each BMU component is
critical for different mechanisms depending on the specific
differentiation stage of the cell. All the signals discussed in the
present review affect the remodeling process. The outcome
depends on the cells generating the stimulus, and the way in
which the stimulus is transferred. Understanding how the events
determining the focal balance between bone resorption and
formation unfold within each BMU provides necessary biological
background information that will help to better identify targets of
therapeutic approaches to osteoporosis.
ACKNOWLEDGEMENTS
We thank Springer Nature Author Services for providing highly professional editing.
ADDITIONAL INFORMATION
Competing interests: The authors declare no competing interests.
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